Primo Unique finds multiple primer pairs, each uniquely amplify one gene in a family.
Primers designed individually for one member of the gene family may also amplify a family member
because of the sequence similarities they share.
Primo Unique inputs multiple sequences for a family,
designs primers for each member, and eliminates those primer
pairs that might amplify a different family member.
To test Primo Unique, click here and copy
the sequences of 15 ribosomal RNAs. Paste the sequences into the Primo Unique
input window, check the "Unique primers" checkbox, and click on "go." Adjust
the 5' and 3' primer regions and repeat.
Primo Pro 3.2 introduces an option for reducing PCR noise by lowering the probability of random
primering on non-target DNA sequences. For example the primer TGCACTACCTGCTGCTGCAC for
the p53 gene looks perfectly OK because it meets most criteria we commonly use for designing
PCR primers. This primer may have high background amplification because the 3’-prime 8 nucleotides
appears in 4800 unique genes in human transcriptome. Primo Pro analyzed in advance
transcriptomes of different species to mark sequences that are over-represented, thus allow users to
select primers with few over-represented sequences at the 3’-end. As a result of reduced random
priming, we expect improved PCR amplification efficiency and cleaner PCR products, especially for
RT-PCR reactions.
Primo Pro 3.2 also introduces a batch mode option for high throughput
PCR primer design. By selecting the batch mode, users can input
multiple sequences and design PCR primers for multiple sequences.
A lot of calculations have been done in advance for Primo Pro.
That is why Primo Pro runs amazingly fast in your web browser.
But to take full advantage of Primo Pro, you would need to install it on your computer.
For example, the online Primo does not allow you to chose a species other than human
because limitations in passing large number of data on the web. Note it searched
only transcribed sequences and each species has a unique pattern of
over-represented sequences. Unlike proteomes of closely related species,
transcriptomes of close relatives usually don't share over-represented
sequences, probably because untranscribed regions are less conserved
and/or many species have young transposons.
The stand-alone version of Primo Pro 3.2 currently has
transcriptome data for 11 species. More species will be added in the near future.
Mac (OSX10): Early version of OSX10 IE5.1 has a bug. Copy and paste will kill the browser.
The bug should have been fixed in later versions of Mac IE.
If you use one of the above browser and you can't run Primo Pro 3.2, please
make sure Java is enabled in your browser. For Internet Explorer, go to
Tools/Internet Options, click on Security Settings, scroll down to
find Microsoft VM, deselect "Disable Java".
Mac OSX 10 IE5.1 has a bug, it does not allow copy/paste into a Java
text field, thus you will not be able to import a new sequence.
Attempting copy/paste may kill your browser. Primo Pro, BioToolKit 160 and PrimerBase 1.2
don't require IE to run, thus don't have this problem.
How to use:
To start using Primo, cut and copy your sequences into the input window.
You may need to use Control-c and Control-v on PC and Apple-c and Apple-v on Mac
to copy and paste.
Numbers and white spaces will be ignored.
Behind the scene:
1. Code for Degenerate Oligos
A A C C
G G T T
U T M (AC)
R (AG) W (AT)
S (CG) Y (CT)
K (GT) V (ACG)
H (ACT) D (AGT)
B (CGT) N (ACGT)
Any other letters will be ignored, so you can paste in a nucleotide
sequences with spaces and numbers.
2. Melting temperature and annealing temperature
Melting temperature is the temperature at which 50% of the oligo and its
perfect complement are in duplex. PCR annealing temperature
a few degree (4-6) lower than the melting temperature is usually used to increase
the probability of primer binding. There are two options for calculating the
melting temperature. The first uses the simple rule of 2 degree for each A or T
and 4 degree for each C or G.
Melting temperature = 4 * Number of G or C + 2 * Number of A or T.
The second "Nearest N" predicts melting temperature using the "Nearest Neighbor" model (Jhon SantaLucia, Proc. Natl. Acad. Sci. Vol. 95, p1460-1465 (1998)).
The cation concentration is assumed to be 50 mM and the primer concentration is assumed
to be 200 nanomolar. The "Nearest N" is presented because it is more
accurate and other formulae can be viewed as approximations of the
"Nearest N".
The "Nearest N" formula has a correction for primer concentration.
If lower or higher primer concentration is used, the rule-of-thumb is
that for each two fold increase or decrease in the primer concentration, the melting temperature should increase or decrease by 1 degree. (Click here to see the relationship between primer concentration and melting temperature.)
For degenerate primers, the effective concentration is lower because of
degeneracy. Users don't need to adjust for the lowered effective concentration if using the "Nearest N" formula, Primo has already taken that into account. Users are suggested to select the "Nearest N"
formula if template sequences consist of degenerate codes.
To compare melting temperatures calculated using the two formulae, type or
copy the primer sequence into the "2nd primer" field, and mouse-click on the
text field. The melting temperature will be calculated using the selected
formula. Click here to see the melting temperatures calculated using both formulae
for some commonly used PCR and sequencing primers.
For degenerate nucleotides, an average is used.
3. Primer-primer dimer
If you select to "Check primer-primer dimers", Primo Pro 3.2 eliminates primers
that might form primer-primer dimers such as the following:
P1: 5'-...GGCGATCG-3'
||||||
3'-GCTAGCGG...-5' : P1 or
P1: 5'....AGGGCCC-3'
|| |
3'-CGCGAAT...-5' : P2
The following single-base pair is also not allowed:
P1: 5'-...AGGTCGCG-3'
|
3'-CGGATT...5' : P2
4. Random background priming
Primo Pro has blacklisted a large number of over-represented sequences of 8 nucleotides by
analyzing transcriptome data from a number of species. Note there will be
a unique list for each species. A primer candidate will be compared to the
blacklist for matches in the last 10 nucleotides. Any one with a match will be
eliminated from the candidate pool. The following three primers will be
eliminated:
Black list oligo: 5'-TGCTGCAC-3'
Primer 1: 5'-TGCACTACCTGCTGCTGCAC-3'
Primer 2: 5'-GCACTACCTGCTGCTGCACC-3'
Primer 2: 5'-GCACTACCTGCTGCTGCACCG-3'
A primer with missmatch at the last two nucleotides is eliminated because of
high probability there will be sequences that match the primer and exonuclease
activities of DNA polymerases.
Batch mode
To use the batch mode, select the batch mode checkbox and input
multiple sequences in the following format:
>seq1 0-100 -100-0
1 ggccgggcgc ggtggctcac gcctgtaatc ccagcacttt gggaggccga ggcgggtgga
61 tcacctgagg tcaggagttc gagaccagcc tggccaacat ggtgaaaccc cgtctctact
121 aaaaatacaa aaattagccg ggcgtggtgg cgggcgcctg taatcccagc tactcgggag
181 gctgaggcag gagaatcgct tgaacccggg aggcggaggt tgcagtgagc cgagatcgcg
241 ccactgcact ccagcctggg caacaagagc gaaactccgt ctcaaaaaaa a
>seq2
1 accgcagcgg acagcgccaa gtgaagcctc gcttccctcc cgcggcgacc agggcccgag
61 ccgagagtag cagttgtagc tacccgccca gaaactagac acaatgtgcg acgaagacga
121 gaccaccgcc ctcgtgtgcg acaatggctc cggcctggtg aaagccggct tcgccgggga
181 tgacgcccct agggccgtgt tcccgtccat cgtgggccgc ccccgacacc agggcgtcat
241 ggtcggtatg ggtcagaaag attcctacgt gggcgacgag gctcagagca agagaggtat
Each sequence starts with an info line with a > sign. The description
of the sequence may be followed by the optional 5' and 3' range for
forward or reverse primers. The three fields are separated by "tab."
If the range value starts with a minus sign, then the counting will be from
the 3'-end of the sequence. If both ranges are missing in the info line,
the input value from the Primo interface will be used. If only one range is present in
the info line, that value will be used for designing forward or reverse primer only. It will be ignored for selecting pairs of forward and reverse primers.
Batch mode is not available in Primo Unique. All other functions of the Primo Pro
is present in Primo Unique.
Get unique primers
Primo Unique first searches for primers for each sequence in the gene family, and then
then checks whether a primer candidate might pair with another sequence in the family.
The 3'-end 8 nucleotides of a primer must not pair to any of the other sequence, i.e., there
must be at least one mismatch in the 3'-end 8 nucleotides for the purposes of
target discrimination.
Species available in the stand-alone version
| Species Name | Common Name |
| Arabidopsis thaliana | Arabidopsis |
| Zea mays | Corn |
| Bos taurus | Cow |
| Drosophila melanogaster | Drosophila |
| Xenopus laevis | Frog |
| Homo sapiens | Human |
| Mus musculus | Mouse |
| Rattus norvegicus | Rat |
| Oryza sativa | Rice |
| Danio rerio | Zebrafish |
Contact us if a species you are interested in is not on this list, we might be able to help.
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